Journal of Experimental Botany, Vol. 53, No. 369, pp. 707-713,
April 1, 2002
© 2002 Oxford University Press
Original Papers |
Regulation of pectic polysaccharide domains in relation to cell development and cell properties in the pea testa
Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK
Received 15 June 2001; Accepted 22 November 2001
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Abstract |
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The occurrence of pectic polysaccharide epitopes in cells and tissues of the pea testa during late stages of seed development have been examined in relation to anatomy and cell properties. Homogalacturonan, in a highly methyl-esterified form, was present throughout late development in all pea testa cell walls, including the thickened cell walls of the outer macrosclereid layer. Two epitopes, characteristic of the side-chains of the rhamnogalacturonan-I domain of pectic polysaccharides, occurred in restricted and separate cell layers of the pea testa. A (1
4)-ß-D-galactan epitope was restricted to regions of the outer cell wall of the testa and to inner regions of the macrosclereid layer at 20 DAA and was absent from the osteosclereid and parenchyma cell walls. By 25 DAA the (14)-ß-D-galactan epitope occurred only in the outer epidermal cell walls. A (15)-
-L-arabinan epitope was also dependent on the developmental stage of the seed and was found with greatest abundance in the walls of the inner parenchyma cells. Cell separation studies indicated that, although calcium cross-links were involved in the maintenance of the link between the macrosclereid layer and proximal cell layers, most cell-to-cell adhesion in the testa was not due to calcium- or ester-based bonds. Key words: Cell properties, pectin, pectic polysaccharides, Pisum sativum L., testa.
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Introduction |
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The testa is of considerable importance to pea seed development. In the early stages, the developing testa plays a role in processing the nutrients that enter the cotyledons for storage and at seed maturity it forms a protective barrier against physical abrasion, insect and microbial attack, and dehydration. The testa consists of a cuticle covering an outer layer of radially-elongated and tightly-adhered macrosclereids, which at maturity are reported to have extensive non-lignified secondary cell walls (Harris, 1983
). The cell layer proximal to the macrosclereids consists of osteosclereids or hourglass cells that, at harvest maturity, have thick non-lignified cell walls separated by extensive intercellular spaces (Harris, 1984). Finally, several layers of starch-containing parenchyma cells make up the inner testa (Harris, 1983, 1984). These diverse tissue layers have distinct properties and functions although these have not been described fully (Angel and Kramer, 1969; Fahloul et al., 1996) and the molecular bases of the distinct properties are unknown. Recent work on pea cotyledon parenchyma cell walls has indicated that the deposition of a pectic galactan component is correlated with an increase in tissue firmness (McCartney et al., 2000). Analyses of the polysaccharide components of the pea testa have been carried out (Ralet et al., 1993a, b; Weightmann et al., 1994, 1995; Renard et al., 1997; Le Goff et al., 2001) but information on individual cell types has been difficult to obtain. It is now timely to assess pectic polysaccharide components of cell walls in the pea testa with the aim of gaining an insight into how pectic polysaccharide structure is regulated in these diverse cell types and how it may impact upon cell and tissue properties.
Three major pectic polysaccharides have been characterized and are thought to occur in all primary plant cell walls. These are homogalacturonan (HG), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) (Albersheim et al., 1996; O'Neill et al., 1990; Mohnen, 1999). HG is a polymer consisting of (14)--linked D-galactosyluronic acid residues and these can be partially methyl-esterified and/or acetylated. The degree of methyl-esterification is highly variable in relation to cells, tissues and within individual cell walls and can greatly influence the pectic network and cell wall properties (Willats et al., 2001). HG is thought to be synthesized and deposited in the cell wall in a highly methyl-esterified form (Mohnen, 1999). In muro de-esterification by pectin methyl esterases results in de-esterified HG which is implicated in cell-to-cell adhesion and other aspects of cell development (Willats et al., 2001). The backbone of RG-I is composed of a repeat of the disaccharide [2)--L-rhamnosyl-(14)--D-galactosyluronic acid-(1] (Lau et al., 1985). Approximately 20–80% of the rhamnose residues are branched with oligosaccharide side-chains of (14)-ß-D-galactans, (15)--L-arabinans or arabinogalactans which can range in size from one to more than 50 glycosyl residues (Albersheim et al., 1996; Mohnen, 1999). The exact structure and function of these neutral side-chains is unclear and the RG-I pectic polysaccharide is highly variable in its structure, its location in cell walls and possible function(s) (Willats et al., 2001). RG-II has a backbone of (14)--linked D-galactosyluronic acid residues with four diverse side-chains and, although structurally complex, appears to be the most conserved of the pectic polysaccharides (Vidal et al., 2000).
The reasons for such complexity of the pectic network are far from clear but are likely to reflect pectic polysaccharide functions in relation to mechanical properties, the maintenance of cell wall matrix porosity and ionic status, cell-to-cell adhesion, and cell expansion. The use of anti-pectin monoclonal antibodies in immunolocalization techniques is an important complement to biochemical knowledge to understand the occurrence and function of pectic polysaccharides (Willats et al., 2001). This report describes the use of a series of anti-pectin monoclonal antibodies to investigate the occurrence of pectic polysaccharides in cell walls during the later stages of the development of the pea testa.
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Materials and methods |
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Plant material
Pea (Pisum sativum L. cv. Avola) seeds were sown in grow-bags (B&Q, UK) and grown at 18 °C in a glasshouse under a regime of 16 h light and 8 h dark. Seedlings were watered daily with tap water; no other nutrient was supplied. Flowers were tagged on the day of full bloom (time of anthesis). Pods were removed at 5 d intervals from 15 d after anthesis (DAA) until 35 DAA.
Monoclonal antibodies
The rat monoclonal antibodies used in this study have all been described previously. JIM5 and JIM7 recognize no/low-methyl-ester and high-methyl-ester epitopes of HG, respectively (Knox et al., 1990; Willats et al., 2000). PAM1 is a phage display monoclonal antibody specific to both unsubstituted and de-esterified stretches or blocks of pectic HG containing in the region of 30 contiguous unesterified GalA residues (Willats et al., 1999). Whole PAM1 phage particles were used in this study. LM5 recognizes an epitope of (14)-ß-D-galactan (Jones et al., 1997). LM6 recognizes an epitope of (15)--L-arabinan (Willats et al., 1998).
Preparation of material for microscopy
Testa material was separated from pea seeds and cut into small cubes of approximately 8 mm3 to include the outer and inner testa. The samples were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) overnight at 4 °C. The samples were then washed for 2 h at 4 °C in three changes of 0.1 M sodium phosphate buffer and dehydrated in an ethanol series consisting of 70% ethanol (2x10 min), 90% ethanol (2x10 min) and 100% ethanol (3x20 min). The samples were then transferred to LR White resin (hard grade acrylic resin, London Resin Co., Basingstoke, UK) and left at 4 °C overnight to allow the resin to infiltrate the tissue. Following two resin changes the samples were placed in gelatine capsules containing LR White resin and polymerized at 60 °C for 24 h. Sections were cut to a thickness of 0.5–1 µm using glass knives on an Ultracut microtome (Reichart-Jung, Austria) and collected on multiwell slides (ICN Biomedicals, Ohio, USA) coated with Vectabond reagent (Vector Laboratories). Sections were blocked for 1 h in PBS/MP and when required de-esterified with 0.05 M Na2CO3 for 30 min and washed three times with PBS prior to blocking. Sections were then incubated with PBS/MP containing a 10-fold dilution of the appropriate rat monoclonal antibody for 1 h. After washing with PBS the sections were incubated with a 100-fold dilution of anti-rat IgG (whole molecule) linked to fluorescein isothiocyanate (FITC; Sigma Chemical Company) in PBS/MP for 1 h, in darkness. After washing with PBS the sections were mounted in a glycerol/PBS-based anti-fade solution (Citifluor AF1, Agar Scientific, UK) and observed on an Olympus BH-2 microscope equipped with epifluorescent optics. In the case of PAM1 the sections were blocked and de-esterified as described above, prior to incubation with PBS/MP containing approximately 1012 phage ml-1 (corresponding to an approximately 1:10 dilution of phage prepared by polyethylene glycol precipitation containing a titre of 1013 phage ml-1, Willats et al., 1999) for 1 h. After washing three times with PBS the sections were fixed with 2.5% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer for 15 min. The sections were then washed three times with PBS and incubated in a 100-fold dilution of anti-M13 monoclonal antibody (Pharmacia Biotech, UK) for 1 h in 3% PBS/MP. Following washing with PBS the sections were incubated with a 100-fold dilution of anti-mouse IgG (whole molecule) linked to fluorescein isothiocyanate (FITC; Sigma, UK) in PBS/MP for 1 h, in darkness. The sections were then washed and mounted as described for rat monoclonal antibodies.
For the cytochemical staining of cellulose the flurochrome Calcofluor White M2R (fluorescent brightner 28, Sigma, UK) was used. A 0.25 µg ml-1 in dH2O was applied to the section for 2 min. After washing extensively with dH2O, the sections were mounted and examined with UV irradiation.
Vortex-induced cell separation of pea testa material
Excised portions of pea testa were extracted with (i) H2O; (ii) 50 mM CDTA, pH 6.5, RT for 16 h; (iii) 50 mM Na2CO3 at 4 °C for 16 h; (iv) 50 mM CDTA at RT for 16 h, 50 mM Na2CO3 at 4 °C for 16 h; (v) 50 mM CDTA at RT for 16 h, 50 mM Na2CO3 at 4 °C for 16 h, 0.5 M KOH at RT for 16 h; (vi) 50 mM Na2CO3 at 4 °C for 16 h, 50 mM CDTA at RT for 16 h; (vii) 50 mM Na2CO3 at 4 °C for 16 h, 50 mM CDTA at RT for 16 h; 50 mM Na2CO3 at 4 °C for 16 h. After each extraction the tendency for cell separation was determined by vortex-induced cell separation (VICS) in a method adapted from Parker and Waldron (Parker and Waldron, 1995). Two sections of pea cotyledon were placed in an Eppendorf tube with 1 ml water. The tissue sections were vortexed for 1 min and the tube was then shaken vigorously ten times. Scores were assigned according to the extent of disruption of the samples.
Freeze-fracture scanning electron microscopy
Fresh and heat-treated (100 °C, 5 min) pea seeds were examined by freeze-fracture scanning electron microscopy (SEM). Pea seeds were frozen in liquid nitrogen and fractured using a pestle and mortar. The samples were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.4) overnight at 4 °C. The samples were then washed for 2 h at 4 °C in three changes of 0.1 M sodium phosphate buffer. Samples were post-fixed in 1% osmium tetroxide in 0.1 M sodium phosphate buffer for 1 h, washed for 15 min as above and dehydrated through 10, 20, 50, 70, 80, 90, and 100% acetone before being critical point dried using liquid CO2. The samples were then mounted, with side of interest uppermost, on to aluminium stubs with silver conducting paint and coated with a layer of gold approximately 25 nm thick. The samples were examined using a CamScan scanning electron microscope (SEM, CamScan, Leica, UK).
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Results |
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Late development of the pea testa
The cellulose-binding fluorochrome Calcofluor White is a useful probe to indicate the cell walls of the pea testa macrosclereid, osteosclereid and parenchyma layers during the later stages of seed development as shown in Fig. 1
. Figure 1a shows a radial section through the pea testa 20 d after anthesis (20 DAA). The thickened cell walls of the outer macrosclereid layer can be clearly seen, in contrast to the cell walls and intercellular spaces of the proximal osteosclereid layer. Proximal to the osteosclereid layer is a layer of parenchyma (12–14 cells in thickness) with the inner and outer parenchyma cell layers being smaller than the central cells (Fig. 1a). During the later stages of pea seed development, the most significant anatomical change to the testa involves the crushing of the smaller parenchyma cells (that form the inner layer of the testa) by the expanding cotyledons to form a region of crushed parenchyma. This progressive event is shown in Fig. 1b–d. The most proximal parenchyma cells are loosely attached by 30 DAA and are often lost during preparation for microscopy.
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Highly methyl-esterified homogalacturonan occurs throughout the pea testa
Anti-HG monoclonal antibodies JIM5, JIM7 and PAM1 were used to characterize the occurrence and methyl-esterification of HG during pea testa development. The specificities of these antibodies have recently been characterized further (Willats et al., 2000). In short, JIM7 binds to a range of HG epitopes with a relatively high level of methyl-esterification, JIM5 binds in preference to HG with a relatively low level of methyl-esterification (but also to fully de-esterified HG) and PAM1 is specific to large de-esterified blocks of HG (Willats et al., 2000). Immunolabelling of resin-embedded sections of pea testa with these anti-HG probes indicated that all cell walls appeared to contain relatively highly methyl-esterified HG. JIM7 bound to all cells and the JIM7 epitope was particularly abundant in the layer of macrosclereids and was less abundant in the most proximal parenchyma cells as shown in Fig. 2a. Most significantly, JIM5 and PAM1 epitopes did not occur abundantly in the sections of testa material although both probes bound to some extent to proximal regions of the macrosclereid layer and the cells of the osteosclereid layer. There was no change in these patterns of occurrence during seed development from 20 DAA to 30 DAA. HG occurred throughout cell walls including the thickened cell walls of the macrosclereid and osteosclereid layers as indicated by JIM7 binding and also by the binding of JIM5 and PAM1 after treatment of sections with sodium carbonate (a treatment that removes methyl-ester groups) as shown in Fig. 2d, f.
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The presence of high ester HG throughout the thickened walls of the macrosclerieds was revealed by labelling of tangential sections through this cell layer with JIM7 as shown in Fig. 3
. Distal regions of the macrosclereid cells have distinctive bands of thickening and more proximal regions have evenly thickened cell walls (Fig. 3). Cellulose, as detected with Calcofluor White, was co-extensive with HG throughout the thickenings (data not shown).
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Changes in (14)-ß-D-galactan and (15)--L-arabinan occurrence during pea testa development
Anti-galactan and anti-arabinan probes LM5 and LM6 were used to characterize the occurrence of RG-I in the testa, these structures being common features of side-chains attached to rhamnose residues in the backbone of RG-I. The galactan and arabinan epitopes had restricted and different occurrences in the testa cell walls. At 20 DAA the (14)-ß-D-galactan epitope was present only in the outer cell wall region of the epidermal macrosclereid layer and the proximal regions of cell walls of the macrosclereid layer and was absent from the osteosclereid and parenchyma cell walls as shown in Fig. 4a. By 25 DAA the (14)-ß-D-galactan epitope was detected only in the outer region of the epidermal cell wall and at a reduced level (Fig. 4b) and was completely absent from sections prepared from 30 DAA testa material (data not shown). At 20 DAA (15)--L-arabinan was detected in low abundance and only in the inner parenchyma cells (Fig. 4c) and by 25 DAA it was abundant throughout the cell walls of the crushed parenchyma (Fig. 4d). By 30 DAA the (15)--L-arabinan epitope was undetected (data not shown). At this developmental stage the crushed parenchyma cells are loosely attached to the parenchyma layer and are lost during sample preparation (Fig. 1
).
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Cell and tissue properties
As an aspect of cell and tissue properties that may relate to pectic polysaccharide occurrence, the cell-to-cell adhesion of pea testa tissues was studied. No tissue or cell separation was observed when excised regions of 25 and 30 DAA testa were incubated in water. Treatment with a calcium chelator (CDTA) resulted in ready separation of the macrosclereid cell layer from proximal cell layers. The tissue cohesion of the two separated regions of the testa were studied further using vortex-induced cell separation (VICS) and extractants known to be capable of solubilizing populations of pectic polysaccharides. Experimental conditions resulting in the separation of the macrosclereid cells (outer testa) are shown in Table 1. The ability of CDTA, and not Na2CO3, to result in some macrosclereid separation indicates that calcium cross-linking may have a role in the maintenance of cell-to-cell adhesion between these cells. Sequential extraction of 25 and 30 DAA material with CDTA/Na2CO3/KOH resulted in a further (but still not complete) separation of the macrosclereid cells. By contrast to the macrosclereid layer, no extractant or combination of extractants resulted in VICS of the parenchyma layer of the testa.
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Cell adhesion in the pea testa was also investigated by examination of the mode of tissue failure using scanning electron microscopy (SEM). Fracture, when frozen, of fresh pea testa material involved rupture across the cell walls, breaking open the cells resulting in the release of the cell contents (Fig. 5a
). By contrast, freeze-fracture of heat-treated pea testa material resulted in cell separation, exposing the outer surface of the cell walls. The effect of heat treatment was most apparent in the osteosclereids and the parenchyma cells (Fig. 5b).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The pea testa can be considered as consisting of four layers: the outer macrosclereid layer with thickened cell walls, a layer of osteosclereids and two parenchyma layers, the inner of which is crushed at maturity. Overall, this structure forms a tough outer covering for the seed and has mechanical properties that are different from the cotyledons that form the bulk of the seed. The HG components of the pectic polysaccharides appear to be a highly methyl-esterified throughout the testa, with low levels of unesterified HG epitopes in proximal regions of the macrosclereid layer. The macrosclereid and osteosclereid layers contain thickened cell walls that have been previously characterized as non-lignified secondary cell walls (Harris, 1983
, 1984). The presence of HG in the thickened cell walls of these cell types demonstrates the diversity of cell wall types in plant systems and that there may not always be clear biochemical distinctions between primary and secondary cell walls. The occurrence of pectin in secondary cell walls of flax fibre cells has recently been reported (Andème-Onzighi et al., 2000).
By contrast to many other organs examined, including the pea cotyledon, pectic galactan and arabinan epitopes, characteristic of the RG-I domain, are not widespread in the pea testa but occur in restricted and distinct locations: (14)-ß-D-galactan is spatially-regulated in the cell walls of the macrosclereid layer and (15)--L-arabinan occurs transiently in the cell walls of the small inner parenchyma cells. The absence of these two epitopes from the parenchyma tissue that forms the bulk of the testa may reflect an absence of RG-I or the presence of structurally-distinct RG-I polysaccharides not recognized by these antibodies. LM5 and LM6 detect two oligosaccharide structures commonly found in RG-I side-chains, but RG-I is potentially a very diverse set of polysaccharides and it is possible that epitopes are masked by substitution (Mohnen, 1999; Willats et al., 2001). The absence of the pectic galactan and arabinan epitopes may also be related in some way to the presence of the highly-methyl-esterified HG in the cell walls. Whatever the reason, pectic components of cell walls of the testa are distinct from the cotyledon and most tissue systems so far examined.
The thin-walled parenchyma cells line the entire inner surface of the pea testa and are known as transfer cells (Tegeder et al., 1999). These cells are also present in broad bean (Vicia faba) seed coats (Offler et al., 1989; Offler and Patrick, 1993). In both these species the transfer cells are thought to be involved in the exchange of sucrose across the extracellular space between the testa and the cotyledons (Weber et al., 1997; Tegeder et al., 1999). In an electron microscopy study of broad bean, plasmodesmata were shown to interconnect all the contiguous cells of the transfer pathway and nearly all plasmodesmata were shown to occur in large pit fields (Offler and Patrick, 1993). The presence of (15)--L-arabinan at the inner face of cell walls in regions surrounding pit fields and the absence of (14)-ß-D-galactan from cell walls in the region of pit fields has been reported in tomato pericarp tissue (Orfila and Knox, 2000). Whether the presence of (15)--L-arabinan in the crushed parenchyma reflects a high level of plasmodesmata or other aspects of the properties of these cells cannot be determined at this stage. Although the functions of pectic galactan and arabinan components are unknown, their localized occurrences described here are suggestive of distinct functions and the observations are an important addition to the growing literature on the spatial and developmental regulation of these two epitopes (Willats et al., 2001).
Pectic polysaccharides are thought to be major contributors to cell adhesion and tissue cohesion. Other than the calcium-based link between the macrosclereid layer and the proximal testa layers (and some calcium cross-linking within the macrosclereid layer), the nature of the bonds maintaining cell adhesion in the pea testa are unknown. The inability of CDTA or saponification to induce cell separation in the parenchyma layer indicates that bonds other than calcium-mediated links and esters are important in cell adhesion in this tissue. This observation reflects the low level of de-esterified HG in this tissue. The SEM studies indicated that heat treatment may be effective in weakening cell-to-cell links in the osteosclereid and parenchyma layers as indicated by fracture planes of frozen material.
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Acknowledgments |
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This work was supported by a BBSRC Unilever Research CASE studentship to LM. We are grateful to Drs Michael J Gidley, Andrew Ormerod and Michael Asquith (Unilever Research) for stimulating discussions and to Adrian Hick for assistance with microscopy.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44(0)1132333144. E-mail: j.p.knox{at}leeds.ac.uk
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Abbreviations |
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DAA, days after anthesis; HG, homogalacturonan; RG, rhamnogalacturonan; SEM, scanning electron microscopy; VICS, vortex-induced cell-separation..
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References |
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H. Konno, T. Nakato, S. Nakashima, and K. Katoh Lygodium japonicum fern accumulates copper in the cell wall pectin J. Exp. Bot., July 1, 2005; 56(417): 1923 - 1931. [Abstract] [Full Text] [PDF]
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E. Leboeuf, S. Thoiron, and M. Lahaye Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths J. Exp. Bot., September 1, 2004; 55(405): 2087 - 2097. [Abstract] [Full Text] [PDF]
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L. M. Fulton and C. S. Cobbett Two {alpha}-L-arabinofuranosidase genes in Arabidopsis thaliana are differentially expressed during vegetative growth and flower development J. Exp. Bot., November 1, 2003; 54(392): 2467 - 2477. [Abstract] [Full Text] [PDF]
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G. Freshour, C. P. Bonin, W.-D. Reiter, P. Albersheim, A. G. Darvill, and M. G. Hahn Distribution of Fucose-Containing Xyloglucans in Cell Walls of the mur1 Mutant of Arabidopsis Plant Physiology, April 1, 2003; 131(4): 1602 - 1612. [Abstract] [Full Text] [PDF]
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